Proteomics is the investigation of gene expression at the protein level. Gene expression and hence cellular protein content can vary both quantitatively and qualitatively according to treatment regimes and/or stress conditions. Protein changes are most easily visualised by separating cellular protein content on polyacrylamide gels (SDS-PAGE) or by using liquid chromatography (RP-HPLC).
In proteomic applications, proteins are separated using these highly resolving techniques and then digested to produce peptides, which can be sequenced using ES-MS/MS or nano LC-MS/MS. Peptide sequences are used to interrogate protein sequence databases in order to identify proteins within the sample. The technique can also be used to identify protein modifications such as phosphorylations, mutations or glycosylation.
This strategy also has other applications; since ES-MS/MS sequencing and database interrogation can detect low levels of a peptide present in a mixture, the technique can be used as part of an impurity profile analysis for recombinant proteins, detecting the presence of minor levels of impurity proteins such as host cell proteins.
The ICH Q6B guidelines, for characterisation and confirmation of biopharmaceuticals in support of new marketing applications, require data on identity, homogeneity and purity, including analyses for product-related impurities focused on separating the desired product from the impurities.